This application is in response to RFA-DA-10-018, Medications Development for Substance Related Disorders (R01). We aim to design, synthesize and test in relevant preclinical models, potential treatments for tobacco addiction. The pharmacological approach proposed is to develop new classes of potent and selective positive allosteric modulators (PAMs) at a5* nicotinic acetylcholine receptors (nAChRs), that may prove efficacious in facilitating smoking cessation efforts. To accomplish this aim, we have assembled a team of highly motivated researchers with expertise in every aspect of the drug discovery process and in animal models of addiction. Under Specific Aim I, we will utilize in vitro cell-based functional assays for a5*nAChRs receptors (and related nAChR subtypes for counter-screens) to drive an iterative medicinal chemistry program aimed at developing new classes of small molecule drugs that amplify a5* nAChR signaling. Specifically, as a primary screening strategy we will examine changes in agonist-induced increases of intracellular calcium signaling via membrane potential dyes and a fluorescent plate reader (FLIPR) in HEK cells stably expressing a5* nAChRs. As a secondary screening strategy, we will assess the effects of novel PAMs on voltage-clamp recordings in Xenopus oocytes expressing a5* nAChRs. Oocyte will provide important insights into the mode of action of PAMs. In addition, we will also test novel a5* nAChR PAMs in a rubidium (86Rb+) efflux assay using synaptosomes from a brain region in which nAChRmediated currents are almost exlusively from a5* nAChRs. Specific Aim II will utilize an iterative medicinal chemistry program based on structure-activity relationships (SAR) to develop new classes of potent and selective a5* nAChR PAMs. Starting points for chemistry will be known pharmacophores from the literature and hits from a HTS campaign. SAR will be employed to optimize new classes of a5* nAChR PAMs for drug metabolism and pharmacokinetics (DMPK), and brain penetration properties. Specific Aim III will test new classes of potent and selective a5* nAChR PAMs with favorable DMPK and brain penetration in the mouse intravenous nicotine self-administration procedure, using wildtype mice and mice with null mutations in a5 nAChR subunits. In addition, the intrinsic rewarding properties of a5* nAChR PAMs and their effects on nicotine reward will be assessed in the brain-stimulation reward (BSR) procedure in rats. This highly integrated multidisciplinary program will capitalize on the unique drug discovery capabilities at Scripps Florida, and promises to yield novel therapeutic entities for smoking cessation.